Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) is a technique that combines the resolving power of high performance liquid chromatography (HPLC) separation with the high accuracy of a mass spectrometer (LC-MS) to achieve high sensitivity separation of analytes using an electrospray ionization (ESI) interface. Mass spectrometry typically involves ionization of chemical compounds to generate charged molecules or molecule fragments for determining the mass to charge ratios. Mass spectrometers remove target components as ions in a gas phase. Following removal, the ions migrate toward the detector of the mass spectrometer under high vacuum.
Liquid chromatography-Mass spectroscopy involves generation of gaseous ions from the liquid eluate exiting the chromatographic separation column. In this regard LC-MS is different from its precursor technology Gas chromatography-Mass Spectroscopy (GCMS) in which the target components, having already been gasified in the GC unit for separation, are introduced directly into the MS unit without further processing. However, if the LC unit were to be connected to the MS unit without an interface, the liquid mobile phase would vaporize and a large amount of gas would be introduced into the mass spectrometer decreasing the vacuum level and preventing the target ions from reaching the detector. Therefore, a major issue in LC-MS was the removal of the mobile phase, and an interface was needed that could convert the eluate of the LC into a reliable source of target ions.
The development of the atmospheric phase ionization (API) technique provided one such interface. Specifically, samples processed using the API technique, are ionized under atmospheric pressure and the solvent or the mobile phase removal takes place outside the vacuum of the spectrometer. Two main types of API interfaces based on different ionization principles are known. They are electrospray ionization (ESI), and atmospheric pressure chemical ionization (APCI). In general, ESI is best suited for ionic compounds with high polarity, and APCI is better suited for low or medium polarity compounds. The principles of ESI and representative ESIMS apparatus are described, for example, in US patents of Smith et al. (U.S. Pat. Nos. 4,842,701, 4,885,706), and Fenn (U.S. Pat. No. 6,297,499) among others, and in the review articles Fenn et al., Science 246, 64 (1989), and Smith et al., Analytical Chemistry 2, 882 (1990) among others.
Some features of the ESI technique are described below to highlight the ESI parameters that influence the sensitivity of detection by LC-ESI-MS. In general, a sample solution is drawn to the tip of a stainless steel capillary which is surrounded by a chamber, commonly referred to as the electrospray chamber. The walls of the electrospray chamber serve as electrodes subjecting the sample to a relatively high voltage of about 3 to 5 kV. The pressure in the chamber is typically maintained at one atmosphere. The capillary is also surrounded by a flow of a nebulizer gas to help generate a spray of the sample. The combination of the electric field and the nebulizer gas causes the liquid emerging from the capillary to be dispersed into a fine spray of charged droplets containing ions of the target. The charged solute molecules or the target ions of the droplets migrate to the surface due to repulsion. As the charged droplets move toward the mass spectrometer, the solvent evaporates causing the droplets to shrink. As a result, charge density in the droplets increase eventually reaching a limit known as the Rayleigh limit. The Raleigh limit is a theoretical limit at which the applied electric field just counterbalances the surface tension of the droplets. See for example, (Wilm M. (2011) Principles of Electrospray Ionization; Mol. Cell. Proteomics. 10(7):M111.009407. As this limit is crossed, the electrostatic repulsion exceeds the surface tension of the solvent and the droplet explodes into smaller droplets. The solvent continues to evaporate and the sequence of evaporation and explosion continues until the droplet becomes so small that even at a charge density below the Raleigh limit, target ions begin to desorb (Ion Desorption Model). According to another proposed mechanism, eventually the droplets become so small that they contain only one target ion which is released when the solvent evaporates (Charge Residue Mechanism).
Electrospray ionization may be carried out in either positive or negative mode. In positive ion mode, the analyte is sprayed at low pH to encourage positive ion formation. In negative ion mode, the analysis is normally carried out well above a molecules isoelectric point to deprotonate the molecule.
For an electrospray ionization interface to function as a reliable source of target ions it is essential that the interface produce a stable spray. Stability is dependent upon a balance between flow rate and applied field. This balance is strongly influenced by solvent properties, particularly the electrical conductivity and surface tension of the solvent. In general, higher conductivity and surface tension require the flow rate to be reduced. And as a result, desirable flow rates for electrospray ionization are found to be in the μL/min range. As a solvent, water is a poor choice for conventional electrospray ionization relative to many common organic solvents (e.g. acetonitrile) because it has both high conductivity and high surface tension. The higher the surface tension the larger the voltage needed for ionization. Therefore, in an LC-ESI-MS analysis, if the water content of the mobile phase is high, or becomes high due to the gradient used, it is difficult to achieve/maintain a stable spray, and consequently, the sensitivity of the analysis deteriorates. However, despite its disadvantages as a solvent, it is often necessary or desirable to use water in the mobile phase to elute polar solutes.